PROJECT SUMMARY/ABSTRACT The long-term objective of this translational U01 is to define pathophysiologically informed and clinically relevant biomarkers based on the sphingolipidomic cargo of extracellular vesicles (EVs) that will serve for the diagnosis and prognosis of subjects with alcoholic hepatitis (AH). AH is the severest form of alcohol-induced liver disease with high mortality, high health care utilization, and the absence of effective therapies. Health care professionals are limited by the lack of predictive and prognostic biomarkers that could risk-stratify AH patients and individualize their therapy based on biomarker profiles. The current proposal links hepatocyte-derived sphinogolipid cargo on EVs to macrophage-mediated liver inflammation by proposing that sphingolipids on EVs from ethanol-damaged hepatocytes recruit macrophages in to the liver, resulting in liver injury and inflammation. Our preliminary data show that EVs are elevated in AH with the following features: i) EV sphingolipids are elevated in AH; ii) EV sphingolipid levels correlate with clinical parameters such as the international normalized ratio, bilirubin, and MELD score; iii) EVs activate proinflammatory macrophage effector responses via sphingolipid signaling; and iv) Hepatocyte-derived EVs can be detected via a novel nanoplasmon enhanced scattering (nPES) assay. This has led to the central hypothesis that circulating EV sphingolipid cargo is a pathogenically relevant biomarker for the diagnosis and prognosis of AH. Therefore, the goals of this proposal are to: i) develop an EV sphingolipidomic diagnostic signature for AH; ii) Demonstrate the ability of EV sphingolipidomics to predict mortality in AH; iii) Extend EV sphingolipidomics to a ?proof-of-mechanism? assay for the beneficial effects observed in AH patients treated with therapeutic agents proposed in our linked complementary Clinical Trial Pilot U01 (RFA-AA-18-005); and iv) Develop a novel clinical test for the diagnosis of AH utilizing nPES technology to diagnose AH by detecting hepatocyte-derived EVs in plasma microsamples. First, we will determine the diagnostic performance of EV sphingolipids for AH in a single-site training cohort followed by a multi-site validation cohort. Second, we will directly test the hypothesis that EV sphingolipids can predict survival and response to a therapy that is expected to reduce the release of EVs from hepatocytes. Third, we will develop and validate a novel clinical assay for the diagnosis of AH. Thus, this multi-PI U01 grant application will yield a liquid biopsy biomarker for AH diagnosis and prognosis.